Name: observation of photobehavior in chlamydomonas reinhardtii
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This study was supported by grants from the Japan Society for the Promotion of Science KAKENHI (https://www.jsps.go.jp/english/index.html) to NU (19K23758, 21K06295), TH (16H06556), and KW (19H03242, 20K21420, 21H00420), from the Ohsumi Frontier Science Foundation (https://www.ofsf.or.jp/en/) to KW, and from the Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials (http://alliance.tagen.tohoku.ac.jp/english/) to NU, TH, and KW.&#160;

In the present study, a wild-type strain of Chlamydomonas reinhardtii, a progeny of the cross CC-124 x CC-125 with agg1+mt-, was used21. CC-124 and CC-125 were obtained from the Chlamydomonas Resource Center (see Table of Materials) and maintained on a Tris-acetate-phosphate (TAP)22, 1.5% agarose medium at 20-25 &#176;C. Any motile strain can be used for this protocol.
1. Cell culture
<ol>
	<li>Culture...

<ol>
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Effects of red background illumination and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea.</a> FEBS Letters. 336 (3), 516-520 (1993).</li><li>Morishita, J., Tokutsu, R., Minagawa, J., Hisabori, T., Wakabayashi, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Chlamydomonas+reinhardtii+mutants+that+exhibit+strong+positive+phototaxis.">Characterization of Chlamydomonas reinhardtii mutants that exhibit strong positive phototaxis.</a> Plants (Basel). 10 (7), (2021).</li><li>Fujiu, K., Nakayama, Y., Yanagisawa, A., Sokabe, M., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+CAV2+encodes+a+voltage-+dependent+calcium+channel+required+for+the+flagellar+waveform+conversion.">Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.</a> Current Biology. 19 (2), 133-139 (2009).</li><li>Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensors+of+ciliary+outer+arm+dynein:+functions+and+phylogenetic+considerations+for+eukaryotic+evolution.">Calcium sensors of ciliary outer arm dynein: functions and phylogenetic considerations for eukaryotic evolution.</a> Cilia. 4 (1), 6 (2015).</li></ol>

The present protocol is easy and not time-consuming. If a C. reinhardtii mutant is suspected of presenting with defects in photoreception or ciliary motion, this method could serve as primary phenotypic analysis.
However, some critical steps exist. One is to use cells in the experiment in the early to mid-log growth phase. After culturing for long periods, cells become less motile, less light-sensitive, and even form palmelloids (cell clumps)27. Another importa...

Light is an indispensable energy source for photosynthetic organisms, but too much light may cause photo-oxidative damage. Thus, phototrophic organisms need to survive under moderate intensity light, where they can photosynthesize but not suffer photo-oxidative damage1. In land plants, chloroplasts cannot move out from the leaf and show photo movements in the cell; chloroplasts move to the periphery of the cell under high light and the cell surface under low light2, whereas many motile algae show photobehaviors that allow them to find proper light conditions for photosynthesis and, thus, facilitate their survival3.
Chlamydomonas reinhardtii is a unicellular green alga regarded as a model organism in research fields like cilia (a.k.a., flagella), photosynthesis, and photobehavior. C. reinhardtii presents with one eyespot and two cilia per cell, used for photoreception and swimming, respectively. The eyespot has two components: channelrhodopsins (ChRs), light-gated ion channels in the plasma membrane, and the carotenoid-rich granule layers located right behind the ChRs. The eyespot acts as a directional light receptor since the carotenoid-rich granule layers function as a light reflector4,5.
ChRs were initially identified as photoreceptors causing photobehaviors in C. reinhardtii6,7,8,9. Although two isoforms, ChR1 and ChR2, are found in the eyespot, knock-down experiments showed that ChR1 is the primary photoreceptor for photobehaviors10. Despite this, ChR2 has received more attention and played a central role in developing optogenetics, a technique to control cell excitation by light11. Therefore, studying the regulatory mechanisms governing photobehaviors in C. reinhardtii will further the understanding of ChR function and improve optogenetics.
After photoreception, C. reinhardtii cells show two types of photobehaviors: phototaxis and photoshock response12. Phototaxis is the behavior of cells swimming in the direction of the light source or the opposite direction, called positive or negative phototaxis, respectively. Photoshock response is a behavior that cells show after sensing a sudden change in light intensity, such as when illuminated by a flash. Cells stop swimming or swim backward (i.e., swimming with the cell body forward) for a short period, typically &#60;1 s.
Ciliary movements in C. reinhardtii are involved in its photobehaviors. Two cilia usually beat like a human&#39;s breaststroke swimming, and this is modulated for photobehaviors. For phototaxis, the forces generated by the two cilia are imbalanced by the modulation of the beating frequency and the waveform amplitude of each cilium13. The cilium closest to the eyespot is called cis cilium, and the other is called trans cilium. These two cilia differ on various points. For example, the ciliary beating frequency of trans cilium in vitro is 30%-40% higher14. In addition, their Ca2+ sensitivity is different. Reactivation of demembranated cell models15 showed that the cis cilium beats more strongly than the trans cilium for Ca2+ &#60;1 x 10&#8722;8 M, while the opposite is true for Ca2+ &#62;1 x 10&#8722;7 M. This asymmetry in Ca2+ sensitivity is possibly important for phototactic turns since mutants lacking this asymmetry do not exhibit normal phototaxis16,17. Conversely, waveform conversion is necessary for photoshock. The ciliary waveform transforms from the asymmetrical waveform in forward swimming to the symmetrical waveform in backward swimming. This waveform conversion is also regulated by Ca2+, at a threshold of 1 x 10&#8722;4 M18,19. Since defects in regulating ciliary movements cause primary ciliary dyskinesia in humans, studying photobehaviors in C. reinhardtii might help in better understanding of these diseases and therapeutic developments20.
Herein, four simple methods to observe photobehaviors in C. reinhardtii are demonstrated. First, a phototaxis assay using Petri dishes is shown, and second, a phototaxis assay against cell suspension droplets. The phenomenon observed in both cases is not strictly phototaxis but photo accumulation, where the cells tend to accumulate close to the light source side or the opposite side. In C. reinhardtii, photo accumulation is mainly caused by phototaxis in a way that can be used as an approximation to phototaxis. Third, a more rigorous assay for phototaxis under a microscope is shown, and last is a photoshock assay under a microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm Cannonball green LED</td>
			<td>Optosupply</td>
			<td>OSPG5161P</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>AC adaptor for the light box</td>
			<td>ATTO</td>
			<td>2196161</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto cell counter</td>
			<td>DeNovix</td>
			<td>CellDrop BF</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Nakalai tesque</td>
			<td>06731-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera flash</td>
			<td>NEWWER</td>
			<td>TT560</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlamydomonas strains CC-124 and CC-125</td>
			<td>Chlamydomonas Resource Center</td>
			<td></td>
			<td>https://www.chlamycollection.org/</td>
		</tr>
		<tr>
			<td>C-mout CCD camera</td>
			<td>Wraymer</td>
			<td>1129HMN1/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop darkroom</td>
			<td>Scientex</td>
			<td>B-S8</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital still camera</td>
			<td>SONY</td>
			<td>RX100II</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dojindo</td>
			<td>G002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Green LED plate</td>
			<td>CCS</td>
			<td>ISLM-150X150-GG</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fujifilm WAKO</td>
			<td>080-01066</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dojindo</td>
			<td>GB70</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nakalai tesque</td>
			<td>238514-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightbox (Flat viewer)</td>
			<td>ATTO</td>
			<td>2196160</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (&#966;3.5 cm)</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium acetate</td>
			<td>Nakalai tesque</td>
			<td>28434-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply for the green LED plate</td>
			<td>CCS</td>
			<td>ISC-201-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Shibuya Optical</td>
			<td>S-RG630</td>
			<td></td>
		</tr>
	</tbody>
</table>

observation of photobehavior in chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Typical C. reinhardtii phototaxis and photoshock response assays are shown here. After cell density estimation, wild-type cell culture (a progeny of the cross CC-124 &#215; CC-125 with agg1+ mt -)23 was washed with photobehavior experimental solution for the phototaxis dish assay. The cell suspension was placed under dim red light for ~1 h. A 2 mL cell suspension was placed in a 3.5 cm Petri dish. The Petri dish was shaken gently, put on a lightbox, and photographed with a camera fixed on...

Watch this Scientific Journal Video about Observation of Photobehavior in Chlamydomonas reinhardtii at JoVE.com

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced ...

Research

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Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

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Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

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Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

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